Kinase inhibition is a viable treatment approach for multiple diseases including cancer, diabetes, and Alzheimer's. Although small molecule inhibitors have occupied center stage in the kinase inhibition arena, challenges with toxicity and emergence of resistance have limited effectiveness and impact. The growing recognition of the need to combine agents to target multiple signaling pathways to treat cancers compounds these already significant challenges. It is imperative that every possible route to developing effective new kinase inhibitors be explored with alacrity. Peptides that interfere with kinase-substrate interactions have the potential to serve as low toxicity, high specificity agents to fill a critical unmet need in this area. We propose to combine multiple innovative technologies to develop and test, in vivo, novel peptide inhibitors of an oncogenic kinase. Using the reverse in-gel kinase assay in combination with novel high-resolution chromatofocusing-based protein separation, naturally occurring Protein Kinase CK2 substrates with the highest observed catalytic efficiency will be identified. Phosphoacceptor sites will be determined by Electron Transfer Dissociation-assisted mass spectrometry, and sites of protein-protein interaction will be probed by Chemical Shift Perturbation-mediated Nuclear Magnetic Resonance spectroscopy. Substrate- competitive pseudosubstrate-class peptide inhibitors, and docking site-class peptide inhibitors will be designed and analyzed for kinase inhibitory activity in vitro and in vivo. For in vivo analyses, kinase inhibitory activity will be monitored using a novel approach to determine the phophorylation state of CK2 and substrates in peripheral blood mononuclear cells after parenteral administration in mice. The successful completion of these aims will provide proof-of-principle that the innovative application of an existing IMAT-funded technology can facilitate the development of new peptide kinase inhibitors to treat cancers and other diseases where aberrant kinase activity underlies disease initiation or progression.